1. Field of the Invention
This invention relates to certain methods and materials for the cloning of DNA and, in particular, to the cloning of "unclonable" DNA using a genetically engineered host cell. This cloning host organism includes particular mutations from existing cells which have been found to stabilize tester DNA plasmids, and can be assembled using standard transducing and plating techniques.
2. Description of Related Art and Introduction to the Invention
The ability to elucidate gene structure and function often depends to a great extent upon the construction of recombinant DNA libraries accurately representing the total genome of a subject organism, followed by the cloning of this recombinant DNA. Generally, the total DNA of cells from the subject organism is isolated and cut into fragments using specific restriction enzymes. The fragmented DNA sequences are then inserted into appropriate vectors for subsequent propagation and amplification in a foreign host. Commonly used vectors include plasmids, cosmids or phages. Examples of host cells suitable for propagation of foreign DNA sequences include bacteria (i.e., E. coli, Bacillus subtilin, Pseudomonas aeruginosa, yeast (i.e., Saccharomyces cerevisiae) Drosophilia (i.e., Drosophilia melongaster) mammalian cell lines (CHO, L-cells) human cell lines (i.e., Hela, Baculovirus, plant cell lines.
To date a number of obstacles have limited the establishment or stable inheritance of foreign (non-native) DNA in host cells. One barrier to establishment of foreign DNA is the presence of restriction endonuclease that cleave such DNA. See, e.g., Bickle, T. (1982) In: Nucleases (Linn, S. M., and Roberts, R. J., eds.). Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; Raleigh, E. A., and Wilson, G. (1986) Proc. Natl. Acad. Sci. USA 83:9070; and Heitman, J., and Model, P. (1987) J. Bact. 169:3243. Genetic inactivation of these restriction enzymes in certain host cells (restriction minus strains) can result in a more efficient introduction of foreign DNA via transformation, transduction, electroporation or conjugation.
Despite such genetic manipulation of host cells, however, many DNA fragments, particularly those from eucaryotic organisms, have been deemed "unclonable." Many of these "unclonable" DNA segments are known to contain sequences capable of forming non-standard secondary and tertiary structures. See, e.g., Erlich, D. (1989) In: Mobile DNA (Berg, D. E. and Howe, M. M., eds.). ASM Publications, Washington D.C. and Santella, R. M., Grunberger, D., Weinstein, I. B., and Rich, A. (1981) Proc. Natl. Acad. Sci. USA 78:1451. DNA that is capable of forming cruciforms (hydrogen bonded hairpin structures formed from inverted repeat sequences) and Z-DNA (a left handed zig zag configured DNA resulting from alternating purine-pyrimidine residues), for example, is rapidly deleted or rearranged in E. coli. See, e.g., Santella, R. M., Grunberger, D., Weinstein, I. B., and Rich, A. (1981) Proc. Natl. Acad. Sci. USA 78:1451 and Fuchs, R. P. P., Freund, A. M., and Bichara, M. (1988) In: Methods and Consequences of DNA Damage Processing (Friedberg, E. C. and Hanawalt, P. C., eds.). Alan R. Liss, Inc., N.Y.
Previous attempts to increase the stability of cloning such complex DNA structures has met with only limited success. See Wyman, A., Wertman, K., Barker, D., Helms, C., and Petri, W. (1986) Gene 49:263-271. Wyman et al. reported that certain mutations in E. coli host cells resulted in an increased fidelity of representation of certain complex eucaryotic DNA sequences. Specifically, this group found that the use of E. coli strains with mutations in homologous recombination pathways (recB, recC, and sbcB, or recD) increased the representation of polymorphic sequences of genomic DNA. While these authors did not fully characterize the polymorphic nature of the DNA sequences cloned, the subject sequences were thought to contain long segments of inverted repetitions or palindromes.
Chalker et al. have reported that the mutation of a single E. coli gene involved in a secondary pathway of homologous recombinations (sbcC) results in the stable propagation of a long palindromic DNA sequence. Chalker, A., Leach, D., and Lloyd, R. (1988) Gene 71:201-205. In marked contrast to the study of Wyman et al., however, the Chalker group did not observe any major effects of mutations in the primary pathway of homologous recombination on palindrome stability. Ishiura et al. studied the effects of mutant strains of E. Coli on the deletion of genomic eucaryotic DNA during cloning. Ishiura, M., Hazumi, N., Koide, T., Uchida, T., and Okada, Y. (1989) J. Bact. 171:1068-1074. These authors report that only a quadruple combination of host mutations (recB, recC, sbcB and either recJ or recN) prevented the deletion of DNA segments during host propagation. Strains with single mutations in either primary or secondary pathways of homologous recombination or combined mutations in recombination and restriction pathways did not prevent deletion. There were no data on the structural configuration of the DNA sequences employed.
The data from these studies, taken together, indicate that host cell mutations of recombination pathways have not lead to increased stability of certain forms of complex DNA during propagation, and the divergent and often conflicting results noted above indicate that, at present, there is not an identifiable or even ascertainable host strain that is suitable for cloning complex, eucaryotic DNA.
The present inventor has discovered that complex DNA sequences can be stabilized during cloning by utilizing hosts with combined mutations in certain nucleic acid recombination and repair pathways. Described herein are novel bacterial hosts designed for cloning foreign DNA by combining DNA repair mutations into host strains deficient in homologous recombination. Mutations which allow for stabilization of tester plasmids containing complex DNA are identified in existing strains and then assembled using standard techniques of transduction, screening, selection and propagation. The resulting bacteria can be used to stabilize and clone complex DNA such as contained in eucaryotic genomes.